Method for recovering silymarin comprising polyhydroxyphenyl chromanones

ABSTRACT

SILYMARIN COMPRISING POLYHYDROXYPHENYL CHROMANONES IS RECOVERED FROM THE DRIED FRUIT OF SILYBUM MARIANUM GAERTN. BY SEPARATING THE FATTY OILS THEREFROM, EXTRACTING THE REMAINING SOLID RESIDUE WITH ETHYL ACETATE, EVAPORATING THE ETHYL ACETATE AND DISSOLVING THE DRY RESIDUE IN A SOLVENT MIXTURE COMPRISING METHANOL, WATER AND PETROLEUM ETHER TO FORM A TWO-PHASE SYSTEM WHEREIN THE CHROMANONES ARE CONTAINED IN THE LOWER PHASE, RECOVERING THE POLYHYDROXYPHENYL CHROMANONES FROM THE LOWER PHASE AFTER SUBJECTING SAME TO MULTIPLE COUNTERCURRENT CONTACT WITH PETROLEUM ETHER.

United States Patent 3,773,932 METHOD FOR RECOVERING SILYMARIN COM- PRISIIgIG POLYHYDROXYPHENYL CHROMA- NON S Rolf Madaus, Cologne-Merheim, Germany, assignor to Dr. Madaus & Co., Cologne, Germany No Drawing. Filed May 27, 1969, Ser. No. 828,353 Claims priority, application Germany, June 1, 1968, P 17 67 666.3; May 6, 1969, P 19 23 082.1 Int. Cl. A61k 27/14 U.S. Cl. 424-195 7 Claims ABSTRACT OF THE DISCLOSURE Silymarin comprising polyhydroxyphenyl chromanones is recovered from the dried fruit of Silybum marianum Gaertn. by separating the fatty oils therefrom, extracting the remaining solid residue with ethyl acetate, evaporating the ethyl acetate and dissolving the dry residue in a solvent mixture comprising methanol, water and petroleum ether to form a two-phase system wherein the chromanones are contained in the lower phase, recovering the polyhydroxyphenyl chromanones from the lower phase after subjecting same to multiple countercurrent contact with petroleum ether.

This invention relates to a novel and improved process for recovering polyhydroxyphenyl chromones. More particularly this invention relates to a method for recovering polyhydroxyphenyl chromones having valuable pharmacological properties.

It has been found that certain polyhydroxyphenyl chromones, for example, silymar-in have a detoxifying and protective effect against various toxicants in the body.

The known methods of isolating polyhydroxyphenyl chromones and which have been used heretofore in the laboratory have not been found suitable for application on a large scale. This is because the starting material, i.e., the fruit of Silybum marianum Gaertn. contains up to 30% of fatty oils and other additional but difficultly removable by-products so that only greasy concentrates are obtained which can be further processed only with extreme difiiculty. Adapting the known methods to large scale operation has furthermore not proved practical because of the extremely long times needed for the processing and because of the many solvents which are required to be introduced individually and repeatedly one after the other in sequence in order to effect the removal of the non-active by-products and fatty oils.

All the methods known to date for removing fatty substances such as are herein involved utilizing conventional'extraction procedures take at least 7 days to carry out and even then in most cases have not proved satisfactory.

'In accordance with the invention, it has now been found that it is not necessary to effect a complete defatting, and that a starting material containing at least from to of fatty oils can be satisfactorily used. Such starting materials can be relatively easily and economically obtainedv by subjecting dried Silybum merianum fruit to defatting by application of high pressure thereto, preferably with a screw press such as is conventionally used for treating other oil seeds. Under such treatment to of the fatty oil can be removed without any loss of active material.

When this defatting method is employed the otherwise additional grinding heretofore required is not necessary as the cell walls or membranes are forced open by the high pressure and by the grinding action of the screw press to such an extent that the active substance quickly and completely passes into the extraction agent.

Patented Nov. 20, 1973 However, because of the remaining content of fatty oil an extraction agent must be utilized in which the fatty oils as well as the active substance are readily soluble. In accordance with the invention it has been found that the most suitable solvent for this purpose is ethyl acetate. Ethyl acetate allows for an exhaustive extraction to be carried out in 24 hours or less while the conventional extraction with acetone requires an extraction time of fourteen days or more.

Following the evaporation of the ethyl acetate an oily greasy partially lumpy product is obtained containing approximately 20 to 30% of active substance. In order to avoid repeated treatment with different solvents and in order to obtain a separation of the active substances from the by-products and fatty oils under economically feasible conditions in accordance with the invention a distributing method involving two substantially immiscible liquid phases have been proposed.

The selection of a suitable solvent system for this purpose was not successful prior to the invention because all of the tested phase pairs from the distribution value G of the mixture of the by-products and of the fatty oil and of the distribution value G of the active materials resulted in such low separation factors B that the value of the required distribution stages necessitated an unbearable expenditure for equipment and labor.

For the phase pair methanol/petroleum ether, wherein the petroleum ether has a boiling point of from 40 to 60 0., there was calculated from the distribution values G =0.25 and G =0.038 obtained for an active material concentration of 2% the separation factor being at 6.5 which would have required theoretically for a 70-80% enrichment about 20 distribution stages. However, in actual operation a very strong phase shifting within the system takes place during the distribution and all attempts to lessen the same by addition of water proved successful.

In accordance with the invention, there was found that the ternary system methanol/water/petroleum ether avoids the disadvantages associated with the methanol/ petroleum ether system. The most favorable methanol/ water/petroleum ether mixture has been found to be methanol/water (95:5) as lower phase in equilibrium with pure petroleum ether, wherein the petroleum ether has a boiling point of from 40 to 60 C. The system in equilibrium furthermore had the advantage of an unexpected shifting of the distribution values in favor of the desired purification process for the active substances.

The applicants invention therefore lies in a method for recovering polyhydroxyphenyl chromones which comprises subjecting the dried fruits of Silybum marianum Gaertn. to high mechanical pressure, for example in a screw press, whereby the cell walls are ruptured and freed of the major amount of fatty oils contained therein, exhaustively extracting the remaining solid residue from the press which still contains oil in an amount of 5 to 10% with ethyl acetate, evaporating the ethyl acetate, dissolving the oil-greasy, partially lumpy dry residue thereby obtained and which contains about 20 to 30% of active material to form a concentration thereof of 2 wt. percent in the lower phase of a solvent system consisting of, for example, methanol water, and petroleum ether in order to separate therefrom the undesirable by-products, centrifuging the solvent system for removal therefrom of flaky solid material constituting impurities and undesirable by-products, thereafter subg'ecting the solvent system to a multiplication, preferably uniform distribution by countercurrent contact of the upper and lower phases, and recovering from the lower phase by concentration in vacuo a brownish powder containing 70 to of polyhydroxyphenyl chromones.

In practical operation of the system in accordance with the invention, it was surprisingly found that a stage value n=6 suflices in order to obtain a 7080% enrichment of the active material.

In the multiplicative distribution which may be carried out in stages or uniformly and is preferably conducted in a countercurrent procedure, the lower phase becomes enriched in active material and may be recovered with the lower phase at the discharge end of the apparatus.

As apparatus, there are required for the uniform multiplicative distribution 6 individual stages with for instance each having one centrifugal separator and one emulsifier which are so arranged in sequence that the purified active material continuously runs off with the heavy or lower phase, the upper or lighter phase which is flowed countercurrent to the lower or heavier phase runs ofi at the opposite end of the battery after passing through the 6 emulsifiers and separator stages.

A comparable degree of efiiciency is obtained using a column device as described by Ziehl, in which case a theoretical bottom value of to 12 is to be used (calculated from HTU=height for one transfer unit).

In both methods of operation, it has proved advantageous to make an addition of material at the beginning of the battery as described by Jantzen as well as in the middle according to Van Dyk. The addition is supplied in the form of a 2% solution in the lower phase.

Following the distribution carried out in accordance with the invention there is recovered the purified active materials in the form of a beige-colored dry product by the evaporation of the lower phase. The dry product contains 70-80% silymarin and may be used in this form directly or it may first be subjected to further purification and/or separation procedures.

In a thin-layer chromatographic separation of the resulting silymarin, there may be obtained on polyamide plates using as the separation agent a mixture of chloroformzmethanol:methylethylketon (60:14:26) and a 1% dinitrophenylhydrazine solution, 4 distinctly separated materials I, II, III and IV.

The material having the largest R -value silymarin I was established as to its basic structure. It has the empirical formula C H O and is a 5,7-dihydroxy-2- (3-methoxy-4'-hydroxyphenyl)-chromanone-3-ol which is substituted in the 7-position with a C H O -radical. It has a melting point of 167 C.

The remaining three materials II, III and IV have not as yet had their exact structure determined, but they can be separated by column chromatography and by crystallization. All of them show, as silymarin I, the same effects in pharmacological tests.

Thus it is justified due to the considerable evidence of pharmacological conformity to maintain the designation silymarin for the product of the process of the invention which consists of a mixture of the materials I, II, III and IV and to designate the individual components as silymarin I, silymarin II, silymarin III and silymarin IV.

Silymarin I can be directly separated from the lower phase running off the distribution apparatus by crystallization. For this purpose, the lower phase after treatment with active charcoal is at first only compressed to such an extent that after the removal of the active charcoal on further compressing, silymarin I is separated in crystalline form.

Thus in accordance with the invention, the recovery of polyhydroxyphenyl chromones, and particularly of silymarin and/or its components as set out above, comprises in accordance with the invention the following steps:

(1) Pressing oil from the Silybum marianum fruit the main amount of fatty oils under high pressure, preferably in a screw press;

(2) Exhaustive extraction of the oil press residue which remains and which still contains some fatty oils with 4 ethyl acetate, evaporation of the solvent and recovery of an oily-greasy primary extract;

(3) Uniform multiplicative distribution of the primary extract in the solvent system according to the invention and the recovery of a dry product having a content of 70 to of silymarin; and

(4) If desired, prior separation of silymarin I by crystallization from the lower or heavier phase and the recovery of the other polyhydroxyphenyl chromones from the remaining mother liquor.

The following example is given to'illustrate the present invention, however, is in nowise to be construed as a limitation thereof.

EXAMPLE (1) 100 kg. of the dried fruits of Silybum marianum were freed from the main amount of fatty oils contained therein using a screw press as conventionally used for oil seeds under high pressure of from 250 to 280 kg./cm. There were thereby obtained approximately 75 to 80 kg. of press residue having an oil content of 5 to 10%.

(2) 80 kg. of this press residue were exhaustively extracted with ethyl acetate. The residue was thereby substantially freed from polyhydroxy phenol chromones and from the residual oil; 80 kilograms of the press residue was treated with 1600 liters of ethyl acetate in a Soxhlet apparatus at room temperature for 24 hours. After evaporating off the ethyl acetate, there was recovered approximately 5 to 6 kg. of an oil-greasy, partly lumpy dry residue having an active substance content of 20 to 30%. The active substance content was determined by the method of Horhammer, L. and Wagner, H., Deutsche Apothekerzeitung, 102, 759 (1962).

(3) The dry residue was dissolved to 2 wt. percent in the lower phase of a system brought into equilibrium comprising methanol/water :5 )/petroleum ether (wherein the petroleum ether has a boiling point of from 40 to 60 C.) and centrifuged off from flaky solid substances. The final volume amounted to about 300 liters of lower phase.

Six individual centrifugal separators adapted for liquid/ liquid-separation were arranged in series with one emulsifying stage, inserted between each two centrifugers, so that in operation the lower or heavier phase passed through the upper phase in counter flow and wherein in each instance in the emulsifying stage both phases were emulsified one into the other so as to effect exchange of the active material. In each following separator stage, the emulsion was again separated into heavy and light phases. The conduits or lines were so arranged that the separated upper phase which was being flowed in countercurrent to the lower phase passed into each preceding emulsifying stage, etc.

The upper and lower phases were first passed continuously against one anotherin the distribution battery containing no active substances, i.e., without loading, so that the phase equilibrium was established. A 2% solution of active material was then fed continuously into the flowing lower phase. On introducing the solution of active material at the inlet of the lower phase, it must be kept in mind that the total flowing volume ratio, i.e., upper phase:lower phase=1:1 must not be changed.

The flow velocity of both phases depends extensively on the degree of efficiency of the emulsifying units and on the separators. The optimum regulation can be ascertained gravimetrically by quantitative determinations of the transition values of the active material.

The lower phase (about 300 l.) discharged from the distribution battery was dried in vacuo at 20 mm. Hg and resulted in a biege-colored to brownish powder in a weight yield of 3.1 kg. The content of active material (silymarin) amounted to between 70 and 80%, with a yield of active material of 2.2%, calculated on the starting material.

(4) Modification for the direct recovery of silymarin I from the lower phase:

The lower phase (about 300 l.) leaving the distribution battery was collected, treated with 1.5 kg. of active charcoal and compressed to 100 l. in vacuum at 20 mm. Hg. The solution while still hot was purified on a filter press and afterwards further compressed. In the range of about 50 to 30 1. remaining volume, an increasing crystallization is initiated. Depending on the degree of concentration, a white to ochre-colored crystallizate was obtained.

The crystallizate had a melting point of 167 to 169 C. It consisted of silymarin I and had a degree of purity of 95 to 100%.

The mother liquor remaining after separating out the crystallizate, which mother liquor still contain silymarin II, III, and IV, was dried by further compressing in vacuum at 20 mm. Hg.

The polyhydroxyphenyl chromones obtained according to the method of the invention demonstrated in experimental tests carried out on animals a marked protective effect against liver-damaging influences of various toxicants.

The degree of liver damage, for instance produced with carbon tetrachloride, can be determined for example by the hexobarbital period of sleep. Inasmuch as damaged liver cells cannot rapidly catabolize the hexobarbital, the period of sleep is very appreciably prolonged in the case of liver damage. When silymarin was administered previous to the hexobarbital, there resulted a shortening of this period of sleep prolongation by 59.2%, i.e., an antagonization of the effect of the carbon tetrachloride by the silymarin.

A liver damaged by carbon tetrachloride furthermore is able to catabolize p-oxyphenyl pyroracemic acid only to a diminished extent, which for this reason therefore appears in increased amounts in the urine. Untreated rats excreted within four hours 10% of the amounts of poxyphenyl pyroracemic acid given, while rats damaged by carbon tetrachloride excreted If, however, they had previously received silymarin, the excretion time normalized at 810%. The animals behaved as if there had not been any liver damage by the carbon tetrachloride.

When the allyl alcohol test according to Eger was carried out, silymarin caused a lessening of the necrosis area by almost half.

In experiments with a-amanitine (poisoning with a fungus of the genus Amanita), the protective etfect of the silymarin was demonstrated by the lessening of the death rate or lengthening of the survival time, by the slower weight decrease, and the more rapid commencement of a weight increase and finally by the distinctive antagonization of the increase of the concentration of the sorbital dehydrogenase.

The cirrhotic liver damage resulting from administration of thioacetamide over a long period of time could also be counter-acted by the simultaneous administration of silymarin. Depending on the silymarin dosage, a considerable inhibition of the body weight loss and an appreciable prolongation of the survival time were obtained.

This effect of the silymarin in protecting the cell structure and the cytometabolism was also confirmed in numerous further experiments. Even damage which had already occurred could be reversed by administering silymarin. It was established by electron-microscopic examination that the structure of the mitochondria and of the endoplasmatic reticulum considerably destroyed after administering wamanitine could be fully restored again by silymarin. The restoration of the disturbed integrity means that silymarin does not only have a protective but also a stabilizing effect.

Thus, the immediate eifect on the structural elements of the liver cell provides for a completely novel form of therapy.

What is claimed is:

1. Process for recovering silymarin comprising polyhydroxyphenyl chromanones which comprises (1) subjecting the dried fruits of Silybum marianum Gaertn. to high mechanical pressure, whereby the cell walls of the fruits are ruptured and the major amount of fatty oils contained in the fruits is separated therefrom, (2) exhaustively extracting the remaining solid residue which still contains fatty oils in an amount of about 5 to 10% with ethyl acetate, (3) evaporating the ethyl acetate, (4) dissolving the oilgreasy, partially lumpy dry residue thereby obtained in a solvent mixture comprising methanol, water and petroleum ether whereby an upper phase and a lower phase are formed and wherein said chromanones are contained in the lower phase, (5) centrifuging said solvent mixture to remove any solid material present therein and separating the upper and lower phases, (6) thereafter subjecting the liquid portion comprising the lower phase to multiple countercurrent contact with petroleum ether, whereby a thorough mixing and separation of both phases occurs, and (7) recovering the polyhydroxyphenyl chromanones from the lower phase by evaporation of the solvent to dryness.

2. Process according to claim 1, wherein the methanol in said solvent mixture contains water in an amount of about 5 parts of water per parts of methanol.

3. Process according to claim 1, wherein the multiple countercurrent contact is carried out in a distribution battery consisting of at least six centrifugal separators and at least six emulsifiers arranged in sequence and wherein the methanol phase is introduced at one end of the battery and petroleum ether is introduced at the opposite end of the battery.

4. Process according to claim 3, wherein the upper phase separated in each of said centrifugal separators is passed into the next preceding emulsifier for mixing therein with the lower phase.

5. Process as claimed in claim 1, wherein the residue obtained in step (2) after extraction with ethyl acetate contains from about 20 to 30% of silymarin comprising polyhydroxyphenyl chromanones.

6. Process as claimed in claim 1, wherein the volume ratio between the said lower phase and the said upper phase in step (6) is about 1: 1.

7. Silymarin comprising polyhydroxyphenyl chromanones as produced by the process claimed in claim 1.

References Cited Chem. Abst., vol. 69 (1968), p. 582301. Chem. Abst. vol. 69 (1968), p. 96396g.

SAM ROSEN, Primary Examiner 

